Phytochemical analysis for ten Peruvian Mentheae (Lamiaceae) by liquid chromatography associated with high resolution mass spectrometry

The profile of secondary metabolites in ten members of tribe Mentheae (Nepetoideae, Lamiaceae) from Peru by liquid chromatography associated with high resolution mass spectrometry, is presented. Salvianolic acids and their precursors were found, particularly rosmarinic acid, caffeic acid ester derivatives, as well as a diversity of free and glycosylated flavonoids as main substances. At all, 111 structures were tentatively identified.

The tropical Andes are considered one of the most diverse areas on the planet in terms of vascular plants. The flora of Perú is extremely rich, and its territory is home to some 25,000 species, almost 10% of all plants in the world. However, the percentage of them scientifically studied is quite low 1 . Phytochemical research on Peruvian biodiversity proved to be fundamental in the development of modern medicine, e.g. the isolation of cocaine from Erythroxylum coca was a milestone in the development of local anesthetics 2 , similarly the isolation of the first antimalarial agent, quinine from Cinchona ledgeriana cortex initiated "the alkaloids golden age" 3 . Most of those phytochemical investigations were conducted overseas, a fact that reflects the absence or the restricted access of resources and infrastructure for developing classical phytochemical research in Peru. Today, modern platforms maybe applied for the metabolic characterization of Peruvian flora, a task that can be achieved by a liquid chromatography associated with high resolution mass spectrometry (LC-HRMS) method since it is less time consuming compared to classic methods of isolation and structure identification. Some recent investigations that exemplify the use of LC-HRMS for describing the phytochemical profile of Peruvian flora include the metabolic profile on medicinal plants of the genus Chuquiraga (Asteraceae) 4 and that related to Capsicum (Solanaceae) fruits 5 .
Perú has several traditional medicine systems, that of the northern Andes 6,7 , that of the southern Andes 8 and that of the Amazonian forest 9 , each one of them with its main and minor plants and particular practices. With the passage of time, those traditional medicines are getting combined a fact that is especially noticeable in Lima city, the capital of Peru 10 . One aspect that is worth to highlight is that, especially in Andean medicines, but not in Amazonian ones, there is an important contribution of plants belonging to the Lamiaceae family to the traditional medicine systems.
The large family Lamiaceae has twelve subfamilies. The Nepetoideae subfamily, with 3400 species and 105 genera, has three tribes 11 : Elsholtzieae, Ocimeae and Mentheae, the latter with 65 genera. The Mentheae tribe is chemically characterized by having volatile terpenoids and a phenolic acid called rosmarinic acid that makes these plants aromatic and with medicinal properties 12,13 Mentheae can also be classified into 3 subtribes: Menthinae (43 genera), Salviinae (10 genera) and Nepetiinae (12 genera) 14,15 . In Peru (Herbario Nacional Universidad de San Marcos-Perú, October 2017), the main genera of Mentheae were Clinopodium (29 species), Hedeoma (1 specie), Lepechinia (11 species), Minthostachys (7 species) and Salvia (60 species www.nature.com/scientificreports/ related to essential oils 16 . In a previous work 17 the contents of rosmarinic acid, triterpenic acids, oleanolic and ursolic were quantified in thirteen Peruvian Mentheae. The highest content of rosmarinic acid was observed in Lepechina meyenii (Walp.) Epling and the highest content of triterpenic acids in Clinopodium revolutum (Ruiz & Pavón) Govaerts. Subsequently 18 , the non-volatile compounds were unambiguously or reasonably identified in two Lepechinia species: L. meyenii and L. floribunda (Benth.) Epling, by LC-HRMS, where the presence of salvianolic acids and diterpenoids were notable. LC-HRMS methods have been used to comprehensively analyze the phenolic components of plants, this implies procedures for the systematic manually identification of mass spectra 19,20 and also the use of suitable software 21,22 , in both cases the procedure involves recording of diagnostic ions for classification and then the identification of characteristic ionic products and neutral losses for confirmation. In the present communication, the profile of secondary metabolites by LC-HRMS is reported for ten Peruvian Mentheae: Clinopodium (4 species), Salvia (4 species), Hedeoma (1 species) and Minthostachys (1 species).

Discussion
This is the first time that the phytochemical profile has been obtained for the ten Peruvian Mentheae (Lamiaceae) here reported. The botanical genera studied were Salvia (Salviinae), Clinopodium, Hedeoma and Minthostachys (Menthinae). While Salvia and Clinopodium are genera of worldwide distribution, Hedeoma and Minthostachys are American and South American genera, respectively. All Salvia species in this work belong to the Salvia subgenus Calosphace Benth. (Epling) 63 . Assignments were based on the search for diagnostic ions, characteristic product ions and neutral losses 19,20,25,40,41 . The fragmentation patterns shown in said references are particularly useful for this work since they are specifically directed to Lamiaceae/Mentheae. The phytochemical profiles of those Mentheae here surveyed are quite similar to their European and Asian relatives. All the species analyzed show the presence of rosmarinic acid, while, quinic acid, 3,4-dihydroxyphenyl-lactic acid "danshensu", protocatechuic aldehyde and caffeic acid are present in most of the samples. Monocaffeoylquinic acids, also called chlorogenic acids, are also frequent but better expressed in Minthostachys. Dicaffeoylquinic acid was detected only in Clinopodium revolutum. All samples contained flavonoids with more diversity in Minthostachys and Clinopodium. Flavonoid-free aglycones predominate in several plants: In Salvia sagitatta, cirsimaritin is abundant 64 , while eupatorin predominates in Clinopodium revolutum 50 , genkwanin in Salvia haenkei 36 and hesperetin in Clinopodium pulchellum 27       www.nature.com/scientificreports/

Data availability
The datasets used and/or analyses during the current study are available from the corresponding author on reasonable request.